Iron is essential for life and crucial to the human central nervous system (CNS). It is fundamental in biochemical pathways including oxidative phosphorylation, myelin synthesis, neurotransmitter production and the synthesis/metabolism of serotonin and dopamine, all of which are essential for normal CNS function. Since iron is highly reactive towards oxygen in aerobic cells, maintenance of cellular iron homeostasis is vital for normal neuronal function and for overall organism viability. Unfortunately, a growing number of human neurodegenerative disorders (Alzheimer's, Huntington's, Parkinson's, Friedreich's ataxia, etc.) have phenotypes confirming disease association to disruption of iron homeostasis. The long-range goal of the Stemmler laboratory is to characterize the functional role proteins play in regulating cellular iron homeostasis, with special interest regarding iron-sulfur cluster (ISC) bioassembly. In eukaryotes, the mitochondrial ISC assembly pathway provides the bulk of the iron-sulfur (Fe-S) clusters used ubiquitously throughout all cells. In yeast, this pathway proceeds through a coordinated effort between the assembly scaffold protein (Isu1/2), a cysteine desulfurase (Nfs1) that provides sulfur, an accessory protein (Isd11) important in Nfs1 stabilization, an adrenodoxin (Yah1) that may provide reducing equivalents to stabilize sulfide for transfer and the iron chaperone frataxin (Yfh1). The objective of this application is to provid molecular and mechanistic details regarding interactions between these key proteins during Fe-S cluster assembly. Our central hypothesis is frataxin plays a direct role in mitochondrial Fe-S cluster assembly by serving as an iron chaperone to Isu and as a modulator of Nfs activity when bound in a stable multiprotein complex that includes Nfs/Isd11/Isu and Yah. Here we will explore the molecular details of frataxin's interaction with Isu, characterize frataxin's interactin with the Nfs/Isd11 complex which modulates cysteine desulfurase activity, and study the structural and dynamic nature of the macromolecular complex that drives Fe-S cluster assembly. PUBLIC HEALTH RELEVANCE: Many neurodegenerative disorders show a correlation between iron accumulation in the central nervous system and iron induced oxidative damage in nerve cells. Alzheimer's, Huntington's, Parkinson's and Friedreich's ataxia are four prevalent neurodegenerative disorders associated with disruption in iron homeostasis, the effect of which leads to the disease state. This proposal is relevant to public health in that it will provide the molecular and biochemical details of the structure and function of key proteins in the iron regulation pathway that are deficient in patients afflicted with disorders related to improper iron sulfur cluster biosynthesis. )